Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-Infectives and Abiotic ...

16 Pages·2017·0.43 MB·English

Article
Ureidopyrazine Derivatives: Synthesis and Biological
Evaluation as Anti-Infectives and Abiotic Elicitors
Ghada Bouz *, Martin Juhás, Pavlína Niklová, Ondřej Janďourek, Pavla Paterová, Jiří Janoušek,
Lenka Tůmová, Zuzana Kovalíková, Petr Kastner, Martin Doležal and Jan Zitko *
Faculty of Pharmacy in Hradec Kralove, Charles University, Heyrovskeho 1203, Hradec Kralove 50005,
Czech Republic; [email protected] (M.J.); [email protected] (P.N.); [email protected] (O.J.);
[email protected] (P.P.); [email protected] (J.J.); [email protected] (L.T.);
[email protected] (Z.K.); [email protected] (P.K.); [email protected] (M.D.)
* Correspondence: [email protected] (G.B.); [email protected] (J.Z.);
Tel.: [phone] (G.B.); [phone] (J.Z.)
Received: 29 September 2017; Accepted: 20 October 2017; Published: 23 October 2017
Abstract: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly
infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to
discover new drugs effective against Mtb. One research area is the synthesis and evaluation of
pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and
biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting
alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions
were performed in pressurized vials using a CEM Discover microwave reactor with a focused field.
Purity and chemical structures of products were assessed, and the final compounds were tested in
vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)
pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-
methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 μg/mL, 18.91 μM) had
high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line.
Therefore 4 and 6 are suitable for further structural modifications that might improve their biological
activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-
4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for
similar activity as abiotic elicitors.
Keywords: abiotic elicitors; anti-infectives; callus culture; ester; Mycobacterium tuberculosis; pyrazinoic
acid; ureidopyrazine

1. Introduction
Tuberculosis (TB) is a common infection that had been successfully treated with appropriate first
line anti-TB drugs, including isoniazid (INH), rifampicin, pyrazinamide (PZA), and ethambutol [1].
Yet in the last few years, this curable infection has become a frequently deadly illness due to the
raising issue of antimicrobial resistance (AMR) [1]. AMR includes multi-drug resistant TB (MDR-TB),
when the bacteria is resistant to both INH and rifampicin, and extensively drug resistant TB (XDR-TB),
when the bacteria is resistant to INH, rifampicin, fluoroquinolones, and one of the three parenteral
second line drugs (amikacin, kanamycin, or capreomycin) [2]. Despite the fact that the annual number
of deaths due to TB has fallen from the year 2000 till today, TB remains one of the top ten causes of
death worldwide and the leading cause of death from infectious diseases [3]. According to the World
Health Organization (WHO) annual report, there were 10.4 million new cases of TB worldwide in
2015, out of which 480,000 cases were MDR-TB [3]. The number of deaths attributed to TB in that year
was 1.4 million, whether due to inaccessibility to treatment or treatment failure [2]. It is estimated
Molecules 2017, 22, 1797; doi:10.3390/molecules22101797 www.mdpi.com/journal/molecules
Molecules 2017, 22, 1797 2 of 16
that by 2050 deaths due to AMR will reach 10 million deaths/year in comparison to 8.2 million
deaths/year due to all types of cancer combined if no measures are implemented to stop resistance
spread [4]. In 2015 alone, the number of deaths of TB due to AMR was 250,000 [3]. The serious issue
of AMR has driven efforts worldwide to find new therapeutic drugs Mycobacterium tuberculosis (Mtb)
bacteria are sensitive to. An ongoing research area is the synthesis and evaluation of PZA derivatives
as potential anti-TB drugs [5–10]. PZA is considered to be an analogue of nicotinamide and its
chemical structure is closely related to INH (Figure 1). PZA plays an important role in conventional
(drug-sensitive) TB treatment regimen as it shortens the duration of therapy from 9–12 months to 6
months [11]. It is converted to its active form, pyrazinoic acid (POA, Figure 1), intracellularly in acidic
pH by a hydrolytic enzyme known as pyrazinamidase (PZase) encoded by the Mtb bacterium itself [12].
Several new specific mechanisms were identified by which PZA or POA exerts its anti-TB activity.
Those include interference with ribosomal protein S1 (RpsA) [12], inhibition of quinolinic acid
phosphoribosyl transferase (QAPRTase) [13], inhibition of aspartate decarboxylase [14], and inhibition
of Fatty acid synthase I (FAS I) [15,16]. The knowledge of such new targets will help to design new
potentially active PZA derivatives that may overcome the issue of AMR.

Figure 1. The chemical structure of (a) INH; (b) nicotinamide; (c) PZA; and POA (d).
In this paper, we focus on the design, synthesis, and anti-infective evaluation of ureidopyrazine
derivatives. In general, urea derivatives have shown wide range of pharmacological activities, including
hypoglycemic, anti-cancer, anticonvulsant, antiviral, and antimicrobial activities, as reviewed
elsewhere [17]. For instance, N-alkylurea hydroxamic acids showed potent antibacterial activity
against both Gram-positive and Gram-negative bacteria by inhibiting peptide deformylase (PDF) that
is essential for bacterial growth [18]. Furthermore, one compound from this series (compound 19) has
been already proved to be a Chk-1 kinase inhibitor with anticancer activity [19], yet have not been
evaluated for any anti-infective property. Title compounds of this study were evaluated for their
antimycobacterial, antibacterial, and antifungal activities in vitro. Two of the prepared compounds,
8 and 18, were further assessed as plant abiotic elicitors since similar pyrazinecarboxylic acid derivatives
were proved to exert such activity. The chemical structures of the latter two compounds also resemble
that of a commercially available plant growth promotor, 1-(2-chloropyridin-4-yl)-3-phenylurea (for its
structure readers may refer to Section 2.2.5).
2. Results and Discussion
2.1. Chemistry
2.1.1. Compounds 1–9
The starting 5-aminopyrazine-2-carboxylic acid (1) was prepared by reacting 5-chloropyrazine-
2-carboxylic acid with an aqueous solution of ammonia to replace the chlorine atom with amino
group (Scheme 1, step a). Then, it was esterified (Fischer esterification) with propanol in the presence
of catalytic amounts of concentrated sulfuric acid to yield propyl 5-aminopyrazine-2-carboxylate (2)
(Scheme 1, step b). The obtained ester was then reacted with six different aromatic substituted
isocyanates in hexane (Scheme 1, step c), resulting in six different aryl substituted ureidopyrazine
propyl esters (Group B). The esterification step preceded the introduction of the urea moiety to the
molecule in order to prevent potential unwanted side-reactions between the isocyanates and the free
carboxylic acid moiety that results in amide formation [20], and also to prevent possible decarboxylation
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at the high reaction temperature. The most biologically active ester 4 was then hydrolyzed to 5 in
methanol and potassium carbonate as a base to compare the biological activity of the ester to the
corresponding free carboxylic acid.

Scheme 1. Synthetic procedures of compounds 1–9. Reagents and Conditions: (a) NH3 (25% aq.sol), MW:
100 °C, 30 min, 80 W; (b) esterification with propanol, H2SO4, MW: 100 °C, 1 h, 80 W; (c) hexane, MW:
120 °C, 1 h, 80 W.
2.1.2. Compounds 10–20
Aminopyrazine was reacted with five different alkyl substituted isocyanates (Group C) and six
different substituted phenyl or benzyl isocyanates (Group D) in one step reaction, in the presence of
hexane as solvent, to yield the corresponding ureidopyrazine derivatives lacking the ester moiety of
group B compounds (Scheme 2). We also attempted reacting 3-chloropyrazin-2-amine and 6-chloro-
pyrazin-2-amine with different isocyanates under same conditions but no product was detected.

Scheme 2. Synthetic procedure of compounds 10–20. Reagents and Conditions: hexane, MW: 120 °C, 1 h, 80 W.
All chemical reactions were performed in pressurized test vials in a CEM Discover microwave
reactor with a focused field. The over-pressurized system of microwave test tubes is essential to
achieve higher temperatures than the boiling points of solvents (propanol and hexane). Moreover,
the closed system prevents the escape of ammonia from the reaction mixture.
Final ureido derivatives were purified using flash chromatography. They were isolated as solids
compounds of white to yellow color, in yields ranging from 12% to 70% of chromatographically pure
products. Then, they were characterized by their melting points, 1H- and 13C-NMR spectra, IR
spectroscopy, and elemental analysis. The acquired analytical data fully supported the corresponding
proposed structures.
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2.2. Biological Activity
2.2.1. Antimycobacterial Activity Evaluation against Mycobacterium tuberculosis,
Mycobacterium kansasii, and Mycobacterium avium
All prepared compounds, including the starting acid and ester that lack the urea moiety, were
screened for in vitro activity against Mtb H37Rv, Mycobacterium kansasii (M. kansasii) and Mycobacterium
avium (M. avium) using a Microplate Alamar Blue Assay [21]. Antimycobacterial activity results were
expressed as minimum inhibitory concentration (MIC) in μg·mL−1 in comparison with INH as
standard (Table 1).
Table 1. Structure of prepared compounds, antimycobacterial activity expressed by minimum inhibitory
concentrations (MIC), and cytotoxicity expressed by IC50 values.
A B C & D

Antimycobacterial Activity MIC (μg/mL)
No. R log P
Mtb M. kansasii M. avium
1 H −0.75 >100 >100 >100
A
2 propyl 0.34 >100 >100 >100
3 benzyl 1.69 >100 >100 >100
4 phenyl 1.62 1.56 >100 >100
5 a phenyl 0.54 >100 >100 >100
B 6 4-methoxyphenyl 1.50 6.25 25 >100
7 2-chlorophenyl 2.18 >100 >100 >100
8 4-chlorophenyl 2.18 25 >100 >100
9 3,4-dichlorophenyl 2.74 >100 >100 >100
10 propyl −0.28 >100 >100 >100
11 butyl 0.13 >100 >100 >100
C 12 pentyl 0.55 100 >100 >100
13 octyl 1.8 25 25 >100
14 decyl 2.64 >100 >100 >100
15 benzyl 0.62 >100 >100 >100
16 4-methoxyphenyl 0.43 >100 >100 >100
17 2-chlorophenyl 1.11 >100 >100 >100
D
18 4-chlorophenyl 1.11 12.5 >100 >100
19 3,4-dichlorophenyl 1.67 >100 >100 >100
20 2-chlorobenzyl 0.14 >100 >100 >100
PZA −1.31 >100 b >100 >100
INH −0.64 0.2–0.4 6.25–12.5 6.25–12.5
a Free acid form of compound 4 prepared by base catalyzed hydrolysis; b MIC value from testing at
pH = 5.6 (acidic) is 6.25–12.5 μg/mL [22]. The value stated in the table is from testing at pH = 6.6 (neutral).
Five of the twenty prepared compounds had anti-TB activity within the range of tested
concentrations. Based on the results of the biological evaluation (Table 1), we can conclude that in
this series the ester moiety is important for anti-TB activity. When compound 4 (MICMtb = 1.56 μg/mL,
5.19 μM) (log P = 1.62) was hydrolyzed to compound 5 (MICMtb > 100 μg/mL, >387.23 μM)
(log P = 0.54), it lost its biological activity, suggesting that the free carboxylic moiety significantly
reduced the lipophilicity and hence could have impaired penetration through the highly lipophilic
mycobacterial cell wall. Lipophilicity is an important aspect of activity against Mtb. The five active
compounds had log P values ranging from 1.11 to 2.18. Compounds with either lower or higher
lipophilicity had diminished activity. However, lipophilicity is not the only determinant of anti-TB
activity; compounds 7 and 8 share the same log P value, yet compound 8 is active while 7 is not. We
also found that non-substituted phenyl (compound 4) or 4-monosubstituted phenyl (compound 6)
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derivatives of ureidopyrazine esters had higher anti-TB activity than other substituents. Interestingly,
4-chloro substitution on the phenyl core resulted in active compounds in the series with ester moiety
(Compound 8, MICMtb = 25 μg/mL), as well as in the series lacking the ester moiety (compound 18,
MICMtb = 12.5 μg/mL). When comparing the chemical structure and anti-TB activity of compounds 3
and 4, we found that the introduction of a -CH2- bridge resulted in loss of biological activity. Aryl
substituted ureidopyrazine esters (Group B) had better activity than the corresponding non-ester
compounds of the same substituent (Group D). When comparing the non-ester compounds (10–20),
alkyl substituted ureidopyrazines (Group C) had inferior activity to the aryl derivatives (Group D).
Among the aliphatic series (Group C), compound 13 (MICMtb = 25 μg/mL, 99.86 μM) exerted the
highest anti-TB activity. This result is consistent with pervious findings in our research group with
N-Alkyl-3-(alkylamino)-pyrazine-2-carboxamides of 6–8 carbon alkyl chain having the best activity [23].
Since the starting acid and ester had no urea moiety, we can infer that urea is an important
pharmacophore for the activity of such designed compounds. Compounds 4 (MICMtb = 1.56 μg/mL,
5.19 μM) and 6 (MICMtb = 6.25 μg/mL, 18.91 μM) are suitable for further structural modifications that
might improve their anti-TB activity and physicochemical properties. Compound 6 (MICM.kansasii = 25
μg/mL, 75.67 μM) and compound 13 (MICM.kansasii = 25 μg/mL, 89.79 μM) were the only two compounds
with moderate activity against M. kansasii. None of the tested compounds showed activity against
M. avium.
2.2.2. Antimycobacterial Activity Evaluation against Mycobacterium smegmatis and
Mycobacterium aurum
In order to compare the activity of prepared compounds on fast growing mycobacteria to that
on previously mentioned slow growing Mtb, M. kansasii, and M. avium, this complementary screening
was conducted. This test has several advantages since M. smegmatis and M. aurum are avirulent
surrogate organisms [24]. Unlike slow growing mycobacteria that cause serious courses of infection
in humans, M. smegmatis and M. aurum result in soft tissue infections in humans [25–27]. The two
latter mycobacteria also have similar antibiotic susceptibility profile to Mtb [28]. Microplate Alamar
Blue assays with INH, rifampicin, and ciprofloxacin as standards was used in performing this test.
None of the tested compounds exerted activity against the mentioned fast growing mycobacteria up
to the highest tested concentration of 125 μg/mL for compounds 9, 17, 20, 250 μg/mL for compounds
8, 14, 19, and 500 μg/mL for the remaining compounds (Supplementary Materials, Table S1),
suggesting that those compounds active against Mtb might work through a pathway not shared
among all species of mycobacteria.
2.2.3. Antibacterial and Antifungal Activity Evaluation
Most compounds were tested in vitro for their biological activity against eight common bacterial
strains and eight fungal strains of clinical importance using standard methods [29]. The test excluded
compounds 3, 6, 7, 9, and 18 due to precipitation in the testing media upon dilution. None of the
tested compounds exerted antibacterial or antifungal activity up to the highest tested concentrations
of 125 μM for compounds 4, 8, 16, 250 μM for compound 14, and 500 μM for the remaining
compounds μM (Supplementary Materials, Tables S2 and S3).
2.2.4. In Vitro Cytotoxicty Assays
TB treatment regimens are known to carry a risk of hepatotoxicity [30]. Therefore, it is important
when developing new anti TB drugs test them for this crucial side effect. The most active compounds, 4
and 6, were further evaluated for any possible cytotoxicity. For this screening, standard hepatic cell
line HepG2 (hepatocellular carcinoma) was used, and results were expressed by the inhibitory
concentration required to decrease the viability of cell population to 50% (IC50) compared to a control
of 100% cell viability. The used a CellTiter 96 assay is based on the reduction of tetrazolium dye MTS
in living cells to formazan, which is then determined colorimetrically. Due to low solubility at higher
concentrations in cell culture medium, it was difficult to determine the exact IC50 of compounds 4 and 6.
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However, it can be stated that those two compounds were nontoxic at their highest tested concentrations
(Table 2, Figure 2).
Table 2. Cytotoxicity of the tested substances in HepG2 cells.
Compound IC50(μM)
4 >25 * (>16 × MICMtb)
6 >50 * (>8 × MICMtb)
PZA >104 [31]
INH 79 × 103 [31]
* Measurement at higher concentrations was not possible due to the precipitation of the tested
compounds in cell culture medium.

Figure 2. Cytotoxic effect of different concentrations of the tested substances on HepG2 cells.
Data points from precipitated concentrations are not shown.
2.2.5. Plant Growth Regulation Activity Evaluation
Previous studies showed that derivatives of pyrazine-2-carboxylic acid used as elicitors were
able to increase secondary metabolites production in in vitro plant cultures. Compounds 8 and 18
structurally resemble a plant growth regulator, 1-(2-chloropyridin-4-yl)-3-phenylurea (Figure 3). This
chemical promotes cell division and growth, leading to an increase in fruit size. Its use has been
approved in the USA on kiwi and grapes [32]. Based on those two facts, the effect of compounds 8
and 18 on rutin production in Fagopyrum esculentum var. Bamby callus culture was assessed.
O
Cl N Cl N
O N O O O
N N Cl N HN N N HN N
H H H H
a b c
Figure 3. The chemical structure of (a) 1-(2-chloropyridin-4-yl)-3-phenylurea; (b) compound 8; and
(c) compound 18.
Full numerical data regarding the content of rutin in callus cultures of F. esculentum after
treatment with compounds 8 and 18 separately can be found in Supplementary Materials, Table S4.
For compound 8, the highest rutin levels, 0.80 (70.2% increase) and 0.83 (76.6% increase) μg·g−1 DW,
were reached 6 and 12 h respectively post treatment in comparison with control (24 K). Gradual
decrease in rutin content was observed after 48 and 72 h of compound 8 application. On the other
hand, compound 18 resulted in a more significant increase in rutin level 6 h post application (115%
increase) when compared to control (24 K, control after 24 h). After 12 and 24 h of elicitor application,
rutin content is increased only about 12.8% and 21.2%. The two compounds decreased rutin
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production in similar fashion after 48 and 72 h; however, compound 18 further decreased rutin
content in comparison with control (168 K, control after 168 h).
These results indicate that the two proposed elicitors are able to increase rutin production in
callus culture of Fagopyrum esculetum var. Bamby. This increase in rutin production level is mainly
affected by the time of elicitor application. According to literature, secondary metabolite production
in general is affected by various factors, including the type of elicitor, its concentrations, and the time
of administration. In our previous studies we declared the positive effect of various concentrations
of pyrazinecarboxamide derivatives on the flavonoids and flavonolignans production in callus and
suspension cultures of Ononis arvensis and Silybum marianum [33,34] and the current results support
ureidopyrazines as well.
3. Materials and Methods
3.1. General Information
All chemicals were of reagent or higher grade of purity. They were purchased from Sigma-
Aldrich (Steinheim, Germany), unless stated otherwise. Progress of reactions was checked by using
Merck Silica 60 F254 TLC plates (Merck, Darmstadt, Germany) with UV detection using 254 nm
wavelength. Microwave assisted reactions were performed in a CEM Discover microwave reactor
with a focused field (CEM Corporation, Matthews, NC, USA) connected to an Explorer 24 autosampler
(CEM Corporation) and this equipment was running under CEM’s SynergyTM software for setting
and monitoring the conditions of reactions. The temperature of the reaction mixture was monitored
by internal infrared sensor. All obtained products were purified by preparative flash chromatograph
CombiFlash® Rf (Teledyne Isco Inc., Lincoln, NE, USA). The type of elution was gradient, using the
mixture of hexane (LachNer, Neratovice, Czech Republic) and ethyl acetate (Penta, Prague, Czech
Republic) as mobile phase. Silica gel (0.040–0.063 nm, Merck, Darmstadt, Germany) was used as the
stationary phase. NMR spectra were recorded on Varian VNMR S500 (Varian, Palo Alto, CA, USA)
at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts were reported in ppm (δ) and were referred
indirectly to tetramethylsilane via signal of solvent (2.49 for 1H and 39.7 for 13C in DMSO-d6; 7.26 for
1H and 77.2 for 13C in CDCl3). Infrared spectra were recorded with spectrometer FT-IR Nicolet 6700
(Thermo Scientific, Waltham, MA, USA) using attenuated total reflectance (ATR) methodology on
germanium crystal. Elemental analysis was performed on vario MICRO cube Element Analyzer
(Elementar Analysensysteme, Hanau, Germany). All values regarding elemental analyses are given
as percentages. Melting points were determined in open capillary on Stuart SMP30 melting point
apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected. Yields are expressed as
percentages of theoretical yields and refer to the isolated products (chromatographically pure) after
all purification steps. Theoretical lipophilicity parameters log P was calculated by algorithms of
program CS ChemBioDraw Ultra 16.0 (CambridgeSoft, Cambridge, MA, USA).
3.2. Synthesis
3.2.1. Compounds 1 and 2
The starting 5-chloropyrazine-2-carboxylic acid (317 mg, 2 mmol) was converted to 5-
aminopyrazine-2-carboxylic acid (1) by substitution reaction with 25% (m/m) aqueous solution of
ammonia (3 mL). The reaction was carried out 10 mL microwave pressurized vials with stirring
(reaction temperature: 100 °C, reaction time: 30 min, power output: 80 W). The reaction was repeated
20 times to yield reasonable quantity of the starting acid. Once the reaction was completed, the vials
content was put onto Petri dish and heated above a water bath with intermittent stirring until a dry
solid was obtained (ammonium salt of the product). To get the free acid form, the ammonium salt
was dissolved in water and drop-wise acidified with 10% hydrochloric acid to reach pH of 4. The
mixture was then left to cool down in room temperature for 5 min then kept in the fridge for 15 min.
The formed free acid crystals were filtered off by filtration paper with suction and left to dry
overnight. After it was dried, the resulting 5-aminopyrazine-2-carboxylic acid (1) was esterified in
Molecules 2017, 22, 1797 8 of 16
several microwave pressurized vials; 3 mL of anhydrous propanol and 2 drops of concentrated
sulfuric acid were added to 278 mg (2 mmol) of compound 1 in each vial. The esterification was
carried out in microwave reactor (reaction temperature: 100 °C, reaction time: 1 h, power output: 80 W).
The completion of reaction was monitored by TLC in system hexane/ethyl acetate (EtOAc) (1:3). The ester
was then purified by flash chromatography using gradient elution 40 to 100% EtOAc in hexane.
3.2.2. Compounds 3–9
The resulted propyl 5-aminopyrazine-2-carboxylate (2) (360 mg, 2 mmol) was reacted with
different aryl substituted isocyanates (2.2 mmol) in hexane as solvent (3 mL). The reaction was carried
out in 10 mL microwave pressurized vials with stirring (reaction temperature: 120 °C, reaction time:
1 h, power output: 80 W). Reactions were monitored by TLC in system hexane/EtOAc (1:3). The
reaction mixture was adsorbed to silica and then purified by flash chromatography using gradient
elution 0 to 100% EtOAc in hexane.
Compound 4 (130 mg, 0.5 mmol) was hydrolyzed to 5 by base catalyzed hydrolysis, using
anhydrous potassium carbonate (3 g, 20 mmol) as base and methanol (approximately 20 mL) as
solvent. The reaction mixture was stirred and heated to 85 °C under reflux in an oil bath for
approximately 6 h. Potassium carbonate was then filtered off and the mixture was acidified with 10%
hydrochloric acid to reach a pH of 3. The solution was evaporated and the product washed with
water to remove any remnants of potassium carbonate and then left to dry.
3.2.3. Compounds 10–20
In a 10 mL microwave pressurized vial, commercially available aminopyrazine (190 mg, 2 mmol)
was reacted with different aryl/alky substituted isocyanates (2.2 mmol) in hexane as solvent (3 mL)
with a magnetic stirrer. The reaction proceeded in a microwave reactor with stirring (reaction
temperature: 120 °C, reaction time: 1 h, power output: 80 W). Reaction process was monitored by
TLC in system hexane/EtOAc (1:3). The reaction mixture was adsorbed to silica and then purified by
flash chromatography using gradient elution 20 to 100% EtOAc in hexane for alkyl substituted
ureidopyrazines and 0 to 70% EtOAc in hexane for aryl substituted ureidopyrazines. N.B.: Alkyl
substituted ureidopyrazines 10–14 needed some time to solidify after the evaporation of solvents
after the flash chromatography.
3.3. Analytical Data of the Prepared Compounds
5-Aminopyrazine-2-carboxylic acid (1). Light pinkish solid. Yield 70%; m.p. 238–245 °C; IR (ATR-Ge,
cm−1): 3316 (-NH-), 1643 (-C=O), 1621, 1591 (arom.); 1H-NMR (DMSO-d6) δ 12.58 (s, 1H,-COOH), 8.51
(d, J = 1.3 Hz, 1H, arom.), 7.91 (d, J = 1.3 Hz, 1H, arom.), 7.27 (s, 2H, -NH2). 13C-NMR (DMSO-d6) δ
165.8, 157.4, 145.7, 131.9, 130.8. Elemental analysis found: C, 42.82%; H, 3.51%; N, 29.64%. Calculated
for C5H5N3O2 (MW 139.11): C, 43.17%; H, 3.62%; N, 30.21%.
Propyl 5-aminopyrazine-2-carboxylate (2). Yellow solid. Yield 45%; m.p. 135–138 °C; IR (ATR-Ge, cm−1):
3317 (-NH-), 1722 (-C=O), 1661, 1584 (arom.); 1H-NMR (DMSO-d6) δ 8.53 (d, J = 1.3 Hz, 1H, arom.),
7.91 (d, J = 1.3 Hz, 1H, arom.), 7.32 (s, 2H, -NH2), 4.16 (t, J = 6.7 Hz, 2H, -CH2-), 1.73–1.62 (m, 2H, -CH2-),
0.93 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (DMSO-d6) δ 164.4, 157.5, 145.7, 132.3, 130.2, 65.8, 21.8, 10.5.
Elemental analysis found: C, 53.44%; H, 6.26%; N, 22.71%. Calculated for C8H11N3O2 (MW 181.20): C,
53.03%; H, 6.12%; N, 23.19%.
Propyl 5-(3-benzylureido)pyrazine-2-carboxylate (3). White solid. Yield 65%; m.p. 190.5–193.3 °C; IR
(ATR-Ge, cm−1): 3329 (-NH-), 1717 (-C=O, ester), 1705 (-C=O, urea), 1625, 1539, 1471, 1456 (arom.); 1H-
NMR (CDCl3) δ 10.67 (s, 1H, urea), 9.36 (s, 1H, urea), 8.82 (d, J = 1.3 Hz, 1H, pyrazine), 8.54–8.50 (m, 1H,
pyrazine), 7.40–7.28 (m, 4H, arom.), 7.32–7.26 (m, 1H, arom.), 4.62 (d, J = 5.8 Hz, 2H, -OCH2-), 4.43–4.33 (m,
2H, -NCH2-), 1.92–1.78 (m, 2H, -CH2-), 1.04 (t, J = 14.8, 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3)
δ 163.9, 155.8, 151.2, 142.7, 138.9, 138.3, 136.0, 135.7, 128.8, 128.7, 127.5, 127.5, 127.4, 67.6, 67.4, 44.7,
44.0, 22.0, 10.4. Elemental analysis found: C, 60.73%; H, 5.66%; N, 17.58%. Calculated for C16H18N4O3
(MW 314.35): C, 61.14%; H, 5.77%; N, 17.82%.
Molecules 2017, 22, 1797 9 of 16
Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (4). White solid. Yield 17%; m.p. 217.9–219.9 °C; IR (ATR-Ge,
cm−1): 3302 (-NH-), 1721 (-C=O, ester), 1704 (-C=O, urea), 1604, 1557, 1540, 1506, 1499 (arom.); 1H-NMR
(DMSO-d6) δ 10.02 (s, 1H, urea), 9.69 (s, 1H, urea), 9.13–9.08 (m, 1H, pyrazine), 8.90–8.81 (m, 1H,
pyrazine), 7.54–7.49 (m, 1H, arom.), 7.36–7.29 (m, 2H, arom.), 7.06–7.02 (m, 2H, arom.), 4.28–4.19 (m,
2H, -OCH2-), 1.72–1.68 (m, 2H, -CH2-), 0.96 (t, 3H, -CH3).13C-NMR (DMSO-d6) δ 163.6, 151.4, 144.0,
138.6, 136.0, 134.7, 129.1, 123.3, 119.1, 66.6, 21.8, 10.5. Elemental analysis found: C, 60.43%; H, 5.59%;
N, 18.38%. Calculated for C15H16N4O3 (MW 300.32): C, 59.99%; H, 5.37%; N, 18.66%.
Propyl 5-(3-Phenylureido)pyrazine-2-carboxylic acid (5). White solid. Yield 35%; m.p. 228–229.3 °C; IR
(ATR-Ge, cm−1): 3569 (-NH-), 1728 (-C=O, acid), 1685 (-C=O, urea), 1596, 1567, 1552, 1500 (arom.);
1H-NMR (DMSO-d6) δ 13.27 (s, 1H, -COOH), 9.98 (s, 1H, urea), 9.73 (s, 1H, urea), 9.12 (d, J = 1.4 Hz,
1H, pyrazine), 8.87 (d, J = 1.4 Hz, 1H, pyrazine), 7.52 (d, J = 8.0 Hz, 2H, arom.), 7.33 (t, J = 7.8 Hz, 2H,
arom.). 13C-NMR (DMSO-d6) δ 165.1, 151.5, 151.2, 144.0, 138.6, 136.8, 134.4, 129.1, 123.2, 119.1.
Elemental analysis found: C, 62.88%; H, 5.50%; N, 24.16%. Calculated for C12H10N4O3 (MW 258.24): C,
63.15%; H, 5.30%; N, 24.55%.
Propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (6). White solid. Yield 39%; m.p. 189–193 °C;
IR (ATR-Ge, cm−1): 3333 (-NH-), 1718 (-C=O, ester), 1702 (-C=O, urea), 1610, 1546, 1508, 1404 (arom.),
1231(-C-O); 1H-NMR (CDCl3) δ 11.05 (s, 1H, urea), 10.87 (s, 1H, urea), 8.90 (d, J = 1.3 Hz, 1H, pyrazine),
8.56 (s, 1H, pyrazine), 7.54–7.42 (m, 2H, arom.), 6.97–6.84 (m, 2H, arom.), 4.39 (t, J = 6.9 Hz, 2H, -OCH2-),
3.84 (s, 3H, -OCH3), 1.92–1.80 (m, 2H, -CH2-), 1.06 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3) δ 163.8,
156.5, 153.4, 150.8, 142.4, 135.9, 135.8, 130.2, 122.0, 114.3, 67.4, 55.5, 22.0, 10.3. Elemental analysis
found: C, 58.58%; H, 5.62%; N, 16.62%. Calculated for C16H18N4O4 (MW 330.34): C, 58.17%; H, 5.49%;
N, 16.96%.
Propyl 5-(3-(2-chlorophenyl)ureido)pyrazine-2-carboxylate (7). White solid. Yield 28%; m.p. 195–197 °C;
IR (ATR-Ge, cm−1): 3567 (-NH-), 1720 (-C=O, ester), 1689 (-C=O, urea), 1636, 1591, 1549, 1511, 1473
(arom.); 1H-NMR (CDCl3) δ 11.86 (s, 1H, urea), 10.90 (s, 1H, urea), 8.86 (d, J = 1.4 Hz, 1H, pyrazine),
8.52 (d, J = 1.4 Hz, 1H, pyrazine), 8.32 (dd, J = 1.5, 8.3 Hz, 1H, arom.), 7.38 (dd, J = 1.5, 8.0 Hz, 1H,
arom.), 7.31–7.23 (m, 1H, arom.), 7.09–6.99 (m, 1H, arom.), 4.39 (t, J = 6.9 Hz, 2H, -OCH2-), 1.94–1.81
(m, 2H. -CH2-), 1.06 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3) δ 163.7, 153.3, 150.4, 142.4, 136.3, 135.4,
134.9, 129.3, 127.8, 124.6, 123.4, 121.3, 67.5, 22.1, 10.4. Elemental analysis found: C, 54.12%; H, 4.59%;
N, 16.58%. Calculated for C15H15ClN4O3 (MW 334.76): C, 53.82%; H, 4.52%; N, 16.74%.
Propyl 5-(3-(4-chlorophenyl)ureido)pyrazine-2-carboxylate (8). White solid. Yield 57%; m.p. 240.6–243.8
°C; IR (ATR-Ge, cm−1): 3328 (-NH-), 1720 (-C=O, ester), 1704 (-C=O, urea), 1606, 1540, 1493, 1471, 1454
(arom.); 1H-NMR (DMSO-d6) δ 10.17 (s, 1H, urea), 10.02 (s, 1H, urea), 9.11–8.99 (m, 1H, pyrazine),
8.92–8.84 (m, 1H, pyrazine), 7.93 (d, J = 2.5 Hz, 1H, arom.), 7.56 (d, J = 8.8 Hz, 1H, arom.), 7.45 (dd,
J = 8.8, 2.5 Hz, 2H, arom.), 4.26 (t, J = 6.6 Hz, 2H, -OCH2-), 1.78–1.67 (m, 2H, -CH2-), 0.96 (t, J = 7.4 Hz,
3H, -CH3). 13C-NMR (DMSO-d6) δ 163.6, 151.4, 151.3, 143.9, 137.6, 136.2, 134.7, 128.9, 126.9, 120.7, 66.6,
21.7, 10.5. Elemental analysis found: C, 53.39%; H, 4.39%; N, 16.53%. Calculated for C15H15ClN4O3
(MW 334.76): C, 53.82%; H, 4.52%; N, 16.74%.
Propyl 5-(3-(2,3-dichlorophenyl)ureido)pyrazine-2-carboxylate (9). White solid. Yield 70%; m.p. 242.6–
243.9 °C; IR (ATR-Ge, cm−1): 3336 (-NH-), 1705 (-C=O, ester), 1655 (-C=O, urea), 1612, 1537, 1481, 1378
(arom.); 1H-NMR (DMSO-d6) δ 10.17 (s, 1H, urea), 10.02 (s, 1H, urea), 9.11–8.99 (m, 1H, pyrazine),
8.92–8.84 (m, 1H, pyrazine), 7.93 (d, J = 2.5 Hz, 1H, arom.), 7.56 (d, J = 8.8 Hz, 1H, arom.), 7.45 (dd,
J = 8.8, 2.5 Hz, 1H, arom.), 4.26 (t, J = 6.6 Hz, 2H, -OCH2-), 1.78–1.67 (m, 2H, -CH2-), 0.96 (t, J = 7.4 Hz,
3H, -CH3). 13C-NMR (DMSO-d6) δ 163.6, 151.4, 151.0, 143.9, 138.8, 136.3, 134.8, 131.4, 130.9, 124.7, 120.3,
119.3, 66.7, 21.7, 10.5. Elemental analysis found: C, 48.65%; H, 3.56%; N, 14.84%. Calculated for
C15H14Cl2N4O3 (MW 369.20): C, 48.80%; H, 3.82%; N, 15.18%.
1-Propyl-3-(pyrazin-2-yl)urea (10). Light yellow solid. Yield 36%; m.p. 104.6–107.1 °C; IR (ATR-Ge, cm−1):
3274 (-NH-), 2963 (-CH2-), 1694 (-C=O), 1586, 1536, 1499 (arom.); 1H-NMR (CDCl3) δ 10.19 (s, 1H, urea),
8.90 (s, 1H, urea), 8.44 (d, J = 1.4 Hz, 1H, pyrazine), 8.14–8.04 (m, 2H, pyrazine), 3.44–3.33 (m, 2H, -
Molecules 2017, 22, 1797 10 of 16
CH2-), 1.70–1.61 (m, 2H, -CH2-) 0.99 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3) δ 154.5, 153, 139.5,
137.3, 136.1, 41.3, 22.7, 10.9. Elemental analysis found: C, 53.68%; H, 6.72%; N, 30.89%. Calculated for
C8H12N4O (MW 180.21): C, 53.32%; H, 6.71%; N, 31.09%.
1-Butyl-3-(pyrazin-2-yl)urea (11). Yellow-light brown solid. Yield 29%; m.p. 107.8–108.5 °C; IR (ATR-Ge,
cm−1): 3276 (-NH-), 2953 (-CH2-), 1688 (-C=O), 1600, 1553, 1502 (arom.); 1H-NMR (CDCl3) δ 10.16 (s,
1H, urea), 8.91 (s, 1H, urea), 8.42 (d, J = 1.5 Hz, 1H, pyrazine), 8.14–8.04 (m, 2H, pyrazine), 3.45–3.37
(m, 2H, -CH2-), 1.66–1.57 (m, 2H, -CH2-), 1.53–1.33 (m, 2H, -CH2-), 0.97 (t, J = 7.4 Hz, 3H, -CH3). 13C-
NMR (CDCl3) δ 156.2, 145, 139.3, 136.4, 136.3, 39.7, 31.9, 20.1, 13.8. Elemental analysis found: C,
55.34%; H, 7.21%; N, 28.31%. Calculated for C9H14N4O (MW 194.24): C, 55.65%; H, 7.27%; N, 28.85%.
1-Pentyl-3-(pyrazin-2-yl)urea (12). Yellow solid. Yield 51%; m.p. 108–110 °C; IR (ATR-Ge, cm−1): 3258
(-NH-), 2955 (-CH2-), 1686 (-C=O), 1618, 1599, 1501 (arom.); 1H-NMR (CDCl3) δ 10.19 (s, 1H, urea),
8.90 (s, 1H, urea), 8.43 (d, J = 8.2 Hz, 1H, pyrazine), 8.13–8.04 (m, 2H, pyrazine), 3.44–3.36 (m, 2H,
-CH2-), 1.44–1.31 (m, 6H, -C3H6-), 0.92 (t, 3H, -CH3). 13C-NMR (CDCl3) δ 156.2, 150, 139.3, 136.4, 136.3,
40, 29.6, 29.1, 22.3, 14. Elemental analysis found: C, 58.78%; H, 7.95%; N, 26.62%. Calculated for
C10H16N4O (MW 208.27): C, 57.67%; H, 7.74%; N, 26.91%.
1-Octyl-3-(pyrazin-2-yl)urea (13). White solid. Yield 43%; m.p. 115.6–117.2 °C; IR (ATR-Ge, cm−1): 3276
(-NH-), 2929 (-CH2-), 1699 (-C=O), 1552, 1535, 1503 (arom.); 1H-NMR (CDCl3) δ 10.21 (s, 1H, urea),
8.91 (s, 1H, urea), 8.43 (d, J = 2.8 Hz, 1H, pyrazine), 8.11 (d, J = 2.8 Hz, 1H, pyrazine), 8.07 (dd, J = 2.8,
1.4 Hz, 1H, pyrazine), 3.40 (td, J = 7.1, 5.5 Hz, 2H, -CH2-), 1.67–1.57 (m, 4H, -C2H4-), 1.43–1.37 (m, 2H,
-CH2-), 1.40–1.26 (m, 4H, -C2H4-), 1.30–1.21 (m, 2H, -CH2-), 0.91–0.84 (m, 3H, -CH3).13C-NMR (CDCl3)
δ 156.2, 150, 139.3, 136.3, 136.4, 136.3, 40, 31.8, 29.8, 29.2, 29.1, 27, 22.6, 14. Elemental analysis found:
C, 60.39%; H, 8.42%; N, 23.23%. Calculated for C13H22N4O (MW 250.35): C, 60.99%; H, 8.53%; N,
23.71%.
1-Decyl-3-(pyrazin-2-yl)urea (14). Yellow solid. Yield 24%; m.p. 121.3–123.5 °C; IR (ATR-Ge, cm−1): 3270
(-NH-), 2926 (-CH2-), 1693 (-C=O), 1600, 1552, 1504 (arom.); 1H-NMR (CDCl3) δ 10.08 (s, 1H, urea),
8.91 (s, 1H, urea), 8.91 (d, J = 2.9 Hz, 1H, pyrazine), 8.91 (d, J = 2.9 Hz, 1H, pyrazine), 8.09–8.04 (m, 1H,
pyrazine), 3.44–3.36 (m, 2H, -CH2-), 1.36–1.21 (m, 16H, -C8H16-), 0.88 (t, J = 6.9 Hz, 3H, -CH3). 13C-NMR
(CDCl3) 156.1, 150, 139.3, 136.4, 136.2, 40, 31.9, 29.9, 29.6, 29.5, 29.3, 27, 22.6, 14.1. Elemental analysis
found: C, 64.82%; H, 9.37%; N, 19.77%. Calculated for C15H26N4O (MW 278.40): C, 64.71%; H, 9.41%;
N, 20.12%.
1-Benzyl-3-(pyrazin-2-yl)urea (15). Beige solid. Yield 32%; m.p. 176.5–179.3 °C; IR (ATR-Ge, cm−1): 3258
(-NH-), 2895 (-CH2-), 1694 (-C=O), 1566, 1540, 1508, 1476, 1448 (arom.); 1H-NMR (DMSO-d6) δ 9.51 (s,
1H, urea), 8.91 (s, 1H, urea), 8.26–8.11 (m, 2H, pyrazine), 7.93–7.85 (m, 1H, pyrazine), 7.41–7.27 (m,
5H, arom.), 4.46–4.31 (m, 2H, -CH2). 13C-NMR (DMSO-d6) δ 154.8, 150.3, 141.3, 140.1, 137.5, 135.6,
128.9, 127.6, 127.3, 43.2. Elemental analysis found: C, 61.35%; H, 5.30%; N, 24.15%.Calculated for
C12H12N4O (MW 228.26): C, 61.67%; H, 5.19%; N, 23.74%.
1-(4-Methoxyphenyl)-3-(pyrazin-2-yl)urea (16). White solid. Yield 12%; m.p. 225.5–227.4 °C; IR (ATR-
Ge, cm−1): 3023 (-NH-), 1682 (-C=O), 1614, 1597, 1567, 1551, 1513, 1501 (arom.), 1247 (-C-O); 1H-NMR
(DMSO-d6) δ 9.49 (s, 1H, urea), 8.99 (s, 1H, urea), 8.29 (dd, J = 2.7, 1.5 Hz, 1H, pyrazine), 8.22 (d, J = 2.7 Hz,
2H, pyrazine), 7.44–7.36 (m, 2H, arom.), 6.93–6.86 (m, 2H, arom.), 3.72 (s, 3H, -OCH3).13C-NMR
(DMSO-d6) δ155.3, 152.0, 149.7, 141.8, 137.8, 135.4, 131.8, 120.9, 114.3, 55.4. Elemental analysis found:
C, 59.01%; H, 4.95%; N, 22.14%. Calculated for C12H12N4O2 (MW 244.25): C, 60.34%; H, 5.30%; N, 21.97%.
1-(2-Chlorophenyl)-3-(pyrazin-2-yl)urea (17). White solid. Yield 26%; m.p. 137.5–240.9 °C; IR (ATR-Ge, cm−1):
3129 (-NH-), 1699 (-C=O), 1593, 1552, 1509, 1482, 1442 (arom.); 1H-NMR (DMSO-d6) δ 10.35 (s, 1H,
urea), 10.28 (s, 1H, urea), 8.85–8.80 (m, 1H, pyrazine), 8.35–8.30 (m, 2H, pyrazine), 7.52–7.46 (m, 2H,
arom.), 7.36–7.29 (m, 1H, arom.), 7.11–7.04 (m, 1H, arom.). 13C-NMR (DMSO-d6) δ 151.8, 149.3, 141.1,
137.9, 135.7, 135.6, 129.4, 127.8, 124.2, 122.5, 121.7. Elemental analysis found: C, 53.13%; H, 3.65%; N,
22.53%. Calculated for C11H9ClN4O (MW 248.67): C, 53.22%; H, 3.70%; N, 22.17%.